Overview: Most effective vaccines in use today generate protective antigen-specific B cell memory. To be effective, memory B cells must target the right specificity, with sufficiently high affinity and express the appropriate antibody class t provide the host with long-term immune protection. These three cardinal attributes of antigen-specific B cell memory emerge progressively under the cognate guidance of follicular helper T (TFH) cells following initial priming and antigen re-challenge in vivo. While we know a great deal about circulating antibodies, still little is understood about the development of high-affinity clas-specific memory B cells that ultimately provides B cell-mediated immune protection in vivo. It is our long-term goal to unravel the cellular programs and molecular mechanisms that regulate the stepwise development of antigen-specific B cell memory in vivo. Research Focus: The production of high-affinity antigen-specific antibodies is one powerful means of targeting antigen clearance and is thought to be a major mechanism of action for all vaccines in use today. Upon exposure to foreign antigens, individual helper T (TH) cells and B cells are recruited into the immune response based on the individual specificity of their expressed antigen receptors. What follows is a sequential cascade of antigen-specific programming, functional differentiation and developmental re-programming that produces class switched, high-affinity memory B cells in ways that will be examined mechanistically in the current research proposal. Specific Aims: We will use a murine protein vaccination model to evaluate the changes in transcriptional programming that accompany stepwise antigen-specific memory B cell development. We propose that antigen recognition by specific B cells at different stages of development initiates separable transcriptional programs that mediate productive contact with antigen-specific TFH cells and propagate effective B cell memory. Here, we wil test multiple facets of this central hypothesis with particular focus on the regulation of affinity maturation in early germinal centers (SA-1), ongoing clonal evolution upon antigen recall (SA-2) and the programming and maintenance of antibody class (SA-3) as three defining attributes of antigen-specific B cell memory. Our studies seek to reveal B cell intrinsic mechanisms of memory B cel development that could define unique targets and new strategies to enhance the action and efficacy of future vaccines and targeted immunotherapies. PUBLIC HEALTH RELEVANCE: Vaccines are powerful tools of preventative medicine that have helped re-define the quality of public health over the past century; however, we still don't understand how vaccines work. This project focuses on antibody molecules and the cells that produce them as the most essential way vaccines protect people for life. We hope to identify fundamental processes that help these cells make the best protective antibodies they can, so we can then design the safest most effective vaccines for the future.